Next Generation Sequencing Facilitates Quantitative Analysis of MiRNAs in fetal Keratinocytes at different gestational ages

Purpose: Next-generation sequencing technologies allow miRNA detection at an unprecedented sensitivity.Early- to mid-gestational fetal mammalian skin wounds heal rapidly and without scarring. The goals of this study are to obtain the differently expressed miRNAs between late- and mid-gestational fetal Keratinocytes for further studies. Methods: Keratinocytes were obtained from six fetal skin samples dividied into two groups: a mid-gestation group and a late-gestation group. RNA extracted from Keratinocytes was used to prepare a small RNA library for next-generation sequencing (NGS) using an Illumina Genome Analyzer IIx. To uncover potentially novel microRNA (miRNAs), the mirTools 2.0 web server was used to identify candidate novel human miRNAs from the NGS data.UCSC Genome Browser and the Vienna RNAfold web server were used to further validate the novel miRNA candidates. The repeatability of the expression levels detected by NGS was confirmed by real-time quantitative RT-PCR. Results: Using an optimized data analysis workflow, we detected a total of 99,354,224 raw reads from the six samples, 85,252,341 (85.81%) were high-quality reads (= 18nt). After alignment to the human genome (GRCH38), the number of genome-aligned reads was 74,678,115 (87.60% of the high-quality reads). The number of sequence reads that correspond to known miRNAs was 61,587,749 (82.47% of the genome-matching reads), as was determined by perfect sequence matching to the database of known miRNAs (miRBase release 18). After removing the matched non-coding RNAs (Release 10), 8,755,258 reads remained for identification of novel miRNA candidates by mirTools 2.0. The results revealed the existence of 202 novel miRNA candidates and 29 known miRNAs that were not listed in miRBase release 18. Of the 202 potential novel miRNAs, 106 candidates were detected by at least 10 counts, by NGS, indicating that they have a higher probability of being novel human miRNAs. Using Student''s t-test, 173 known miRNAs and 23 novel miRNA candidates were found to be statistically significant (P values < 0.05). The expression of 22 novel miRNA candidates and 88 known miRNAs was changed by more than 2.0-fold (known: 15 up-regulated and 73 down-regulated; novel: two up-regulated and 20 down-regulated). Conclusions: Our study represents the first detailed analysis of human miRNAs in fetal Keratinocytes. Using mirTools 2.0 web server, we have revealed the existence of 202 novel miRNA candidates. Of the 202 potential novel miRNAs, 106 candidates were detected by at least 10 counts, by NGS, indicating that they have a higher probability of being novel human miRNAs. Taken together, our results provide compelling evidence that dynamic expression of miRNAs in fetal Keratinocytes at different gestational ages. MiRNAs presenting altered expression at different gestational ages in fetal Keratinocytes may contribute to scarless wound healing in early- to mid-gestational fetal Keratinocytes, and thus may be new targets for potential scar prevention and reduction therapies. Overall design: small RNA profiles of human fetal Keratinocytes at different gestational ages were generated by next-generation sequencing on a Genome Analyzer (GA) (Illumina) IIx platform

研究目的
下一代测序技术能够以前所未有的灵敏度实现微小RNA（miRNA）的检测。妊娠早中期的哺乳动物胎儿皮肤伤口可快速愈合且无瘢痕形成。本研究旨在获取妊娠晚期与中期胎儿角质形成细胞间差异表达的miRNA，以供后续研究使用。

研究方法
从6份胎儿皮肤样本中分离得到角质形成细胞，分为妊娠中期组与妊娠晚期组两组。提取角质形成细胞中的RNA，利用Illumina Genome Analyzer IIx平台构建小RNA文库，用于下一代测序（next-generation sequencing, NGS）。为发掘潜在的新型miRNA，使用mirTools 2.0在线服务器从NGS数据中筛选候选新型人类miRNA。进一步通过UCSC基因组浏览器与Vienna RNAfold在线服务器验证候选新型miRNA。采用实时定量逆转录聚合酶链反应（real-time quantitative RT-PCR）验证NGS检测到的表达量重复性。

研究结果
通过优化的数据分析流程，我们从6份样本中共检测到99,354,224条原始读段，其中85,252,341条（占优质读段的85.81%）为长度≥18nt的优质读段。将读段比对至人类基因组（GRCH38）后，共获得74,678,115条基因组比对读段（占优质读段的87.60%）。通过与已知miRNA数据库（miRBase第18版）进行完全序列匹配，发现61,587,749条序列读段对应已知miRNA（占基因组匹配读段的82.47%）。去除匹配到的非编码RNA（第10版）后，剩余8,755,258条读段用于通过mirTools 2.0鉴定新型miRNA候选。结果显示存在202个新型miRNA候选以及29个未被miRBase第18版收录的已知miRNA。在202个潜在新型miRNA中，有106个候选通过NGS检测到至少10个计数，提示它们成为新型人类miRNA的概率更高。采用Student氏t检验分析，发现173个已知miRNA与23个新型miRNA候选存在统计学显著性差异（P值<0.05）。其中22个新型miRNA候选与88个已知miRNA的表达量变化幅度超过2.0倍（已知miRNA中15个上调、73个下调；新型miRNA候选中2个上调、20个下调）。

研究结论
本研究首次对胎儿角质形成细胞中的人类miRNA进行了详细分析。通过mirTools 2.0在线服务器，我们发掘出202个新型miRNA候选。在202个潜在新型miRNA中，有106个候选通过NGS检测到至少10个计数，提示它们成为新型人类miRNA的概率更高。综上，我们的研究结果提供了有力证据，证明不同胎龄的胎儿角质形成细胞中miRNA的表达具有动态变化特征。在不同胎龄的胎儿角质形成细胞中表达异常的miRNA，可能参与调控妊娠早中期胎儿角质形成细胞的无瘢痕伤口愈合过程，因此有望成为瘢痕预防与治疗的潜在新靶点。

整体实验设计
通过Illumina Genome Analyzer IIx平台的下一代测序技术，获取不同胎龄人类胎儿角质形成细胞的小RNA表达谱。